The paracellular route is the dominant pathway for solute flow across the intestinal epithelial barrier, and its permeability depends on the regulation of the intercellular tight junction (tj). The tj is a dynamic and complex structure whose physiological regulation remains largely undefined. During the 10 years of funding of this grant, our studies have focused on the mechanisms of action of a protein elaborated by Vibrio cholerae, zonula occludens toxin (Zot). Our experiments using Zot as a tool to gain insights into the regulation of tj function led to the discovery of zonulin, a human eukaryotic Zot analogue, and to the definition of some of its physiological (innate immune mucosal response of the gut) and pathological (autoimmune diseases) roles. We have demonstrated that Zot and zonulin each target the same surface intestinal receptor and activate the same intracellular signaling events that lead to reversible tj disassembly. We have extended our findings to disease states (including celiac disease and type 1 diabetes) characterized by a leaky gut and have established the role of zonulin in their pathogenesis. Our studies on the Zot structure-function analysis, combined with the studies on Zot trafficking within V. cholerae demonstrated that the N-terminal portion of the 12 kDa mature Zot contains a proteinase activated receptor (PAR) activating peptide (AP) motif that causes protein kinase C (PKC)alpha-dependent ZO-1 and myosin 1 beta phosphorylation. This signaling is associated to loss of ZO-1-occludin and ZO-1-claudin 1 protein-protein interaction, increase in ZO-1-myosin 1 beta protein-protein association, and increase in actin polymerization. Our overall hypothesis is that both Zot with its PAR-AP N-terminus and zonulin with its protease activity target PAR-2 expressed on mature enterocytes. This receptor-ligand interaction is coupled to activation of phospholipase C (PLC) and PKC-alpha, actin polymerization, and tj disassembly. We hypothesize that PKCalpha-mediated phosphorylation of scaffold proteins, coupled with actin polymerization temporarily alter the dynamic of protein-protein interactions within the tj multiprotein complex leading to reversible tj disassembly. The long-term objective of this proposal is to use Zot-derived PAR-AP synthetic peptide as a probe to elucidate the intracellular signaling events operative in Zot/zonulin-induced tj disassembly to gain insights into tj regulation at the cellular and molecular levels. PUBLIC HEALTH RELEVANCE: We hypothesize that PKCalpha-mediated phosphorylation of scaffold proteins, coupled with actin polymerization temporarily alter the dynamic of protein-protein interactions within the tj multiprotein complex leading to reversible tj disassembly. The long-term objective of this proposal is to use Zot-derived PAR-AP synthetic peptide as a probe to elucidate the intracellular signaling events operative in Zot/zonulin-induced tj disassembly to gain insights into tj regulation at the cellular and molecular levels.